Recombinant human and animal adenoviruses are used extensively for their application in gene therapy and vaccination. The adenovirus vector is used as a carrier for a gene of interest to be introduced into host cells, for instance, to express a gene or part thereof encoding a desired antigen to elicit an immune response.
More than 50 different human adenovirus serotypes have been identified. Of these, adenovirus serotype 5 (Ad5) has historically been studied most extensively for use as gene carrier. Recently, several other serotypes such as human Ad11, Ad26, Ad34, Ad35, Ad48, Ad49 and Ad50 and simian adenoviruses have been studied as vectors in view of lower levels of pre-existing neutralizing antibodies against these serotypes in the human population (see e.g., WO 00/70071). Promising examples of these are recombinant Ad35 (rAd35) and rAd26, which are studied in clinical trials.
The molecular biology of adenoviruses which possess a double stranded DNA genome of about 34-38 kb has been studied in detail. All adenoviruses are characterized by various inverted terminal repeats (ITRs) of about 100 bp in size (Dan et al., 2001, Virus Genes 22: 175-179; Liu et al., 2003, Curr Top Microbiol Immunol 272: 131-164), which are conserved among the serotypes of the different groups (Shinagawa et al., 1987, Gene 55: 85-93). The genome ends are covalently attached to the terminal protein (TP) at the 5′ ends of the genome. The ITRs harbor the origin of replication (Bernstein et al., 1986, Mol Cell Biol 6: 2115-2124; Challberg & Rawlins, 1984, Proc Natl Acad Sci USA 81: 100-104; Guggenheimer et al., 1984, Proc Natl Acad Sci USA 81: 3069-3073; Harris & Hay, 1988, J Mol Biol 201: 57-67; Hay, 1985, EMBO J 4: 421-426; van Bergen et al., 1983, Nucleic Acids Res 11: 1975-1989; Wang & Pearson, 1985, Nucleic Acids Res 13: 5173-5187) and are crucial for DNA replication, containing binding sites for cellular proteins that promote replication and facilitating panhandle formation. The ITR sequences possess a short highly conserved canonical “core region” that ranges from nucleotide 9-18 (Liu et al., supra). The terminal 8 nucleotides, preceding this core region, however, vary between adenovirus types and isolates (Alestrom et al., 1982, Gene 18: 193-197; Dan et al., supra; Jacobs et al., 2004, J Gen Virol 85: 3361-3366; Purkayastha et al., 2005, J Clin Microbiol 43: 3083-3094; Rademaker et al., 2006, J Gen Virol 87: 553-562; Shinagawa et al., 1987, supra; Shinagawa et al., 1983, Virology 125: 491-495; Shinagawa & Padmanabhan, 1980, Proc Natl Acad Sci USA 77: 3831-3835; Tokunaga et al., 1982, Gene 18: 329-334; Houng et al., 2006, J Clin Virol 35: 381-387). While most adenoviruses display the CATCATCA (nucleotides 1-8 of SEQ ID NO:6) sequence in the terminal 8 nucleotides, several alternative sequences have been described.
The demand for recombinant adenoviruses is raising steeply in view of the variety of diseases that appear amenable for treatment or prophylaxis using these gene transfer vehicles, in combination with the large number of people affected by these diseases and ever increasing population world-wide.
For clinical batches that are intended for administration to humans, large-scale production of Recombinant Adenovirus (rAd) must be safe and efficacious, and comply with Good Manufacturing Practice (GMP) guidelines. One aspect important in this respect, is the homogeneity of such produced rAd batches.